J. Bryan Smith

Metabolism of [14C]arachidonic acid by human platelets

A time dependent incorporation of [1-14C] arachidonic acid into platelet phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine was observed in platelet-rich plasma. When platelets, so labelled, were washed and treated with thrombin, there was a major decrease in the radioactivity of phosphatidylcholine and phosphatidylinositol. This decrease was accounted for by the appearance of several previously identified (Hamberg and Samuelsson (1974) Proc. Natl. Acad. Sci. U.S. 71, 3400) 14C-labelled oxygenated products of arachidonic acid.

Arachidonic Acid Causes Sudden Death in Rabbits

Injection of sodium arachidonate (1.4 milligrams per kilogram) into the marginal ear veins of rabbits caused death within 3 minutes. Histological examination showed platelet thrombi in the microvasculature of the lungs. Rabbits were protected from the lethal effects of arachidonic acid by pretreatment with aspirin. Fatty acids closely related to arachidonic acid did not cause death.

Prostaglandin D2 inhibits the aggregation of human platelets.

Abstract Prostaglandin D2, heretofore considered to be biologically inactive, was found to be more than twice as potent as prostaglandin E1 as an inhibitor of aggregation in human citrated platelet-rich plasma. It was much less potent than prostaglandin E1 in rabbit or rat platelet-rich plasma. This prostaglandin, or a derivative of it, may prove to be of greater value than prostaglandin E1 in maintaining the viability of stored platelets and as an antithrombotic agent.

Selective binding site for [3H]prostacyclin on platelets.

Prostacyclin (PGI(2)) is the most potent, naturally occurring inhibitor of platelet aggregation known. To determine whether PGI(2) is bound by platelets, high specific activity [9-(3)H]PGI(2) was synthesized by iodination and subsequent base treatment of the labeled precursor [9-(3)H]prostaglandin (PG)F(2alpha) methyl ester. Binding experiments were performed at room temperature with normal citrated human platelet-rich plasma that contained [(14)C]sucrose or [(14)C]PGF(1alpha) as an internal marker for the extracellular space. Binding of [(3)H]PGI(2) plateaued within 2 min and this bond radioactivity could be displaced rapidly by excess nonradioactive PGI(2). Scatchard analysis of concentration-dependent binding yielded a hyperbolic plot which appeared to be caused by the existence of two classes of binding sites. The higher affinity class has a dissociation constant of 12.1+/-2.7 nM and a capacity of 93 (+/-21)sites per platelet. The lower affinity class had a dissociation constant of 0.909+/-.236 muM and a capacity of 2,700+/-700 sites per platelet. The relative ability of PGI(2), PGE(1), PGE(2), and 6-keto-PGF(1alpha) to displace [(3)H]PGI(2) initially bound to the higher affinity class of sites were 100:5:<0.3: <0.3. These relative abilities parallel the relative potencies of these compounds as inhibitors of ADP-induced platelet aggregation in vitro. However PGD(2), which is more potent than PGE(1) as an inhibitor of aggregation, did not displace bound [(3)H]PGI(2). The higher affinity binding site for PGI(2) appears to be the specific receptor through which PGI(2) exerts its effect on platelets.

Studies on the protective effect of prostacyclin in acute myocardial ischemia.

The ability of prostacyclin (PGI2) to alter responses to acute myocardial ischemia was studied in open-chest, anesthetized cats. PGI2 was infused intravenously at 0.5 nmoles kg-1 min-1 in cats subjected to 5 h of myocardial ischemia by occlusion of the LAD coronary artery, and in sham-operated controls. GI2 infusion resulted in significantly decreased arterial blood pressure and inhibition of platelet aggregation. Coronary ligation resulted in significant S-T segment elevations lasting 5 h in vehicle-treated animals but only 1 h in cats with myocardial ischemia and receiving PGI2. At 5 h, cats with ischemia and given the vehicle showed S-T segment elevations significantly greater than the other two groups. Ischemic myocardium from vehicle-treated animals exhibited significantly less creatine phosphokinase (CPK) specific activity than normal tissue from the same hearts or myocardial tissue from the other two groups. This loss of CPK from ischemic myocardium of the cats given vehicle was reflected in plasma CPK specific activities which were significantly greater than those of sham-operated cats. The cats with ischemia and treated with PGI2 exhibited lower plasma CPK activities. These changes were moderated by PGI2 infusion during myocardial ischemia. PGI2 infusion may protect the ischemic myocardium by reducing oxygen demand, primarily through reductions in cardiac work, and by perhaps inhibiting platelet aggregation and preserving myocardial cell integrity.

Detection of thromboxane B2 in peripheral blood of patients with Prinzmetal's angina

The plasma levels of thromboxane B2 (TxB2) were determined by radioimmunoassay in 6 consecutive patients with Prinzmetals angina and in 9 healthy volunteers. In the normal group TxB2 was not detectable (= 0.5 pmoles/ml), while in patients with variant angina TxB2 was consistently detected (1.5-140 pmole/ml).

Responsiveness of platelets and coronary arteries from different species to synthetic thromboxane and prostaglandin endoperoxide analogues

1 Platelet‐rich plasma (PRP) from humans, cats, dogs (after addition of 10 μm adrenaline), rabbits and guinea‐pigs aggregated in response to sodium arachidonate or 9,11‐azo‐prostaglandin H2, while PRP obtained from sheep was unresponsive to either agent. 2 The stable thromboxane (Tx) analogues, carbocyclic TxA2 (CTA2) and pinane TxA2 (PTA2) significantly inhibited these aggregatory responses in platelets from humans, dogs and guinea‐pigs, while PTA2 but not CTA2 produced significant inhibition in cat platelets. The aggregatory response of PRP from rabbits was not significantly blocked by either analogue. 3 CTA2 and the endoperoxide analogue 9,11‐methanoepoxy PGH2 (U‐46619) constricted coronary arteries from cats, dogs, rabbits and guinea‐pigs, while sheep vessels were unresponsive to either analogue. 4 Vasoconstrictor responses to U‐46619 were significantly attenuated by PTA2 in vessels from all species. However, constriction produced by CTA2 was blocked significantly only in vessels from cats, dogs and guinea‐pigs. 5 These results demonstrate the species differences which exist in the responsiveness of platelets and coronary arteries to thromboxane and endoperoxide analogues. Furthermore, the results illustrate the importance of species selection in the study of thromboxane antagonists for potential therapeutic use.

Prostaglandin endoperoxides and thromboxane A2 can induce platelet aggregation in the absence of secretion

PLATELET aggregation and secretion are widely studied not only because they are believed to reflect the major in vivo platelet function, formation of the primary haemostatic plug, but also because they involve fundamental cellular regulatory mechanisms. The synthesis of biologically active prostaglandin (PG) intermediates (endoperoxides and thromboxanes) seems to be an important step in the regulation of platelet function1,2, but the physiological roles and mechanism(s) of these compounds are unknown. It is claimed by Malmsten et al.3 and Samuelsson et. al.4 that PG endoperoxides and thromboxane A2 cause platelet aggregation only by inducing platelets to secrete ADP that in turn causes aggregation. In view of observations that platelets deficient in releasable ADP (storage pool-deficient platelets or platelets depleted of storage granules) still aggregate in response to arachidonic acid5–7, the precursor of the intermediates, the validity of this claim has been open to question. We have investigated platelet stimulation by PG endoperoxides (PGG2 and PGH2), an endoperoxide analogue ((15S)-hydroxy-11α, 9α-(epoxymethano) prosta-5Z, 13E-dienoic acid; U-46619), thromboxane A2-like material and arachidonic acid, using a new instrument that simultaneously monitors aggregation and secretion in the same platelet suspension8, permitting careful analysis of the relationships between aggregation and secretion. We report here that although high concentrations of the endoperoxide analogue, the natural endoperoxides and thromboxane A2 induce both platelet aggregation and secretion, low concentrations cause aggregation without detectable secretion.

Antibodies which antagonise the effects of prostacyclin.

ANTIBODIES directed against prostaglandin–albumin conjugates have been used previously as prostaglandin antagonists. Horton and Poyser1 have demonstrated that the oestrus cycle in the guinea pig is prolonged in animals immunised against prostaglandin F2α (PGF2α). Ferreira et al.2 demonstrated that rats containing antibodies to E-type prostaglandins develop a significantly reduced inflammatory oedema in response to carageenin compared with control animals. We report here that antibodies can be used to antagonise the effects of prostacyclin both in vitro and in vivo.

Potent constriction of cat coronary arteries by hydroperoxides of arachidonic acid and its blockade by anti-inflammatory agents

The omega-6 and omega-9 hydroperoxides of arachidonic acid caused dose-dependent constriction of cat coronary arteries in concentrations of 10(-8) to 10(-5) M. Their potency was comparable to that of prostaglandin (PG) E2, and PGF2 alpha and 100 times greater than that of arachidonic acid. The cyclooxygenase inhibitor, meclofenamate markedly reduced constriction caused by the hydroperoxides but potentiated constriction caused by the prostaglandins. The effects of the hydroperoxides were also reduced by indomethacin and dexamethasone but were unaffected by the thromboxane synthetase inhibitor imidazole. Since the hydroperoxides are not substrates for cyclooxygenase, it is suggested that they have a direct effect on the arteries which can be antagonized by anti-inflammatory drugs.